Human immunodeficiency virus (HIV)-1 is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1 entry into target cells is initiated by a high-affinity binding of HIV-1 envelope gp120 glycoprotein to the primary receptor CD4, and the subsequent interaction of CD4-bound gp120 with the appropriate chemokine receptor (co-receptor), either CXCR4 or CCR5. See, e.g., Feng et al. (1996) Science 272:872-877; Deng et al. (1996) Nature 381:661-666. Most HIV strains are dependent upon the CD4/CCR5 receptor/co-receptor combination to gain entry into a cell and are termed CCR5 (or R5) tropic. Some viral strains however are dependent on the CD4/CXCR4 receptor/co-receptor combination and are termed CXCR4 (or X4) tropic, while others can utilize both the CD4/CCR5 and CD4/CXCR4 combinations and are termed dual (or R5/X4) tropic.
Recombinant transcription factors comprising the DNA binding domains from zinc finger proteins (“ZFPs”) or TAL-effector domains (“TALEs”) and engineered nucleases including zinc finger nucleases (“ZFNs”), TALENs, CRISPR/Cas nuclease systems, and homing endonucleases that are all designed to specifically bind to target DNA sites have the ability to regulate gene expression of endogenous genes and are useful in genome engineering and gene therapy, including in the inactivation of HIV receptors such as CCR5 and CXCR4. See, e.g., U.S. Pat. Nos. 9,045,763; 9,005,973; 8,956,828; 8,945,868; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et at (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy. Nuclease-mediated gene therapy can be used to genetically engineer a cell to have one or more inactivated genes and/or to cause that cell to express a product not previously being produced in that cell (e.g., via transgene insertion and/or via correction of an endogenous sequence), thereby improving both the safety and efficiency with which HSC can be engineered. In particular, the use of engineered nucleases such as zinc finger nucleases (ZFNs), TALENs, TtAgo and CRISPR/Cas9 systems, provide the capability of precisely engineering specific genes. The nucleases act by creating double-stranded breaks (DSB) at a targeted DNA sequence, whose subsequent repair is then exploited to achieve one of three outcomes—gene knockout, gene mutation, or the site-specific addition (i.e. insertion or integration) of new genetic material (transgenes or fragments thereof) at the locus. For example, if DSB repair occurs through the error-prone NHEJ pathway, the result can be small insertions and/or deletions of nucleotides at the break site that thereby disrupt an open-reading frame.
Examples of uses of transgene insertion include the insertion of one or more genes encoding one or more novel therapeutic proteins, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild-type gene in a cell containing a mutated gene sequence, and/or insertion of a sequence that encodes a structural nucleic acid such as miRNA or siRNA. Examples of useful applications of mutation or ‘correction’ of an endogenous gene sequence include alterations of disease-associated gene mutations, alterations in sequences encoding splice sites, alterations in regulatory sequences, alterations in sequences to cause a gain-of-function mutation, and/or alterations in sequences to cause a loss-of-function mutation, and targeted alterations of sequences encoding structural characteristics of a protein. Transgene construct(s) is(are) inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, e.g., U.S. Pat. Nos. 9,045,763; 9,005,973; 7,888,121 and 8,703,489.
Clinical trials using these engineered transcription factors and nucleases have shown that these molecules are capable of treating various conditions, including cancers, HIV and/or blood disorders (such as hemoglobinopathies and/or hemophilias). See, e.g., Yu et al. (2006) FASEB J. 20:479-481; Tebas et at (2014) New Eng J Med 370(10):901. Thus, these approaches can be used for the treatment of diseases.
Currently, the availability of highly active antiretroviral therapy (HAART) has transformed HIV infection into a chronic condition. However the associated costs, potential side-effects, and practical challenges of adhering to life-long drug regimens mean that alternative drug-free strategies to control HIV are needed. These include approaches based on genetically modifying cells to be HIV-resistant, either by directly engineering the CD4 T cells that HIV infects, or by targeting precursor cells including hematopoietic stem and progenitor cells (HSC) that give rise to these cells in vivo. See, e.g., U.S. Pat. Nos. 7,951,925 and 8,524,221 which describe nuclease-mediated inactivation of CCR5 in HSC at levels that proved sufficient to fully suppress HIV-1 replication in a humanized mouse model. Targeted integration of anti-HIV fusion proteins is described in U.S. Patent Publication No. 20120093787 and 20130171732.
Nonetheless, there remains a need for additional strategies of providing genetically modified cells for use in treatment and/or prevention of HIV.